Nucleic acid bulge-detecting agent

ABSTRACT

This invention relates to a metal complex of formula (I). The metal complex is capable of cleaving a bulge-containing nucleic acid at the bulge site with high specificity.

This application is a divisional application of U.S. Ser. No. 09/302,334filed Apr. 30, 1999, now U.S. Pat. No. 6,348,588.

BACKGROUND OF THE INVENTION

Nucleic acid bulges refer to regions of unpaired bases in adouble-stranded nucleic acid molecule. These bulges have been known totake part in many important biological processes.

For example, ENA bulges form crucial motifs for specific nucleicacid-protein recognition and binding. It has been known that the humanimmunodeficiency virus transactivator protein Tat binds to athree-pyrimidine bulge in the response element TAR. See, e.g., Weeks etal., Science 249, 1281-1285 (1990). Nucleic acid bulges also produceframeshift mutations which can change the product of the proteintranslation and result in various disorders. According to one report,Myerowitz et al., J. Biol. Chem. 263, 18587-18589 (1988), approximately70% of Ashkenazi Tay-Sachs disease is caused by a four-base pairinsertion mutation in the HEX A gene encoding the α-subunit ofhexosaminidase A. Another disease, cystic fibrosis, is also caused byframeshift mutation. A three-base pair deletion (AF508) is commonlyfound among cystic fibrosis patients. Rommens et al., Am. J. Hum. Genet.46, 395-396 (1990).

Comparative gel electrophoresis assay has been used to detect thepresence of bulges in nucleic acids. This assay differentiates nucleicacids with and without bulges by their different mobility in gel.However, it can only provide information as to whether a nucleic acidcontains a bulge. Thus, there exists a need for a detection method whichcan provide additional information, e.g., the location of a bulge in anucleic acid.

SUMMARY OF THE INVENTION

One aspect of this invention relates to a metal complex of formula (I):

Each of R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸, independently, is hydrogen,alkyl, alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl, amino,aminoalkyl, alkylcarbonylamino, alkylaminocarbonyl, alkylcarbonyl,alkylcarbonylalkyl, alkoxycarbonyl, alkylcarbonyloxy, cycloalkyl,heterocycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl. Each ofR² and R³, and R⁶ and R⁷, independently, optionally join together toform a cyclic moiety which is fused with the two pyridyl rings to whichR² and R³, or R⁶ and R⁷ are bonded. The cyclic moiety, if present, isoptionally substituted with alkyl, alkoxy, hydroxyl, hydroxylalkyl,halo, haloalkyl, amino, aminoalkyl, alkylcarbonylamino,alkylaminocarbonyl, alkylcarbonyl, alkylcarbonylalkyl, alkoxycarbonyl,alkylcarbonyloxy, cycloalkyl, heterocycloalkyl, aryl, aralkyl,heteroaryl, or heteroaralkyl. Each of L¹ and L², independently, is—C(R^(a)) (R^(b))—, —O—, —S—, or —N(R^(c))— and each of R^(a), R^(b),and R^(c), independently, is hydrogen, alkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl. M is aCo, Ni, Ru, Rh, Mn, Os, Ag, Cr, Zn, Cd, Hg, Re, Ir, Pt, or Pd ion, andthe oxidation state of M is 0, 1, 2, 3, or 4. Each of X¹ and X²,independently, is a labile ligand.

Examples of a metal complex of formula (I) includecobalt(II)(hexaazacyclophane)(trifluoroacetate)₂, cobalt (II)(hexaazacyclophane) (H₂O) (trifluoroacetate) ruthenium(II)(hexaazacyclophane) (trifluoroacetate)₂, andmanganese(II)(hexaazacyclophane)(trifluoroacetate)₂.

Another aspect of this invention relates to a method of specificallycleaving a nucleic acid bulge. The method comprising contacting thebulge with a metal complex of formula (I), supra, where M is a Fe, Co,Ni, Ru, Rh, Mn, Os, Ag, Cr, Zn, Cd, Hg, Re, Ir, Pt, or Pd ion. In oneembodiment, the method is performed in the presence of an oxidant, e.g.,hydrogen peroxide, or in a medium having a pH values which ranges from4-9.

In this disclosure, a nucleic acid bulge is a region in adouble-stranded nucleic acid molecule (DNA or RNA), the region having atleast one unpaired nucleotide and being flanked by two pairednucleotides. The nucleic acid bulge can contain 1-5 unpaired nucleotides(e.g., 1-3). Using nucleic acid substrate A in FIG. 1 as an example, thenucleic acid bulge present therein contains three unpaired nucleotides,i.e., T₆, C₇, and T₈. This three-base bulge is flanked by two pairednucleotides, i.e., A₅-T₂₃ and G₉-C₂₂. In contrast, the C₁₃-A₁₈ hairpinloop, which is also present in substrate A, is not a bulge as theunpaired nucleotides are only connected to one paired nucleotide, i.e.,C₁₂-G₁₉. A nucleic acid bulge can also contain two nucleotides. See thebulge present in substrate D which is formed of two unpairednucleotides, i.e., C₆ and T₇.

A salt of a metal complex of formula (I) is also within the scope ofthis invention. Note that a metal complex of formula (I) can bepositively charged. A salt of such a metal complex can be formed with ananionic counterion. Examples of counterions include fluoride, chloride,bromide, iodide, sulfate, sulfite, phosphate, acetate, oxalate, andsuccinate.

As described above, each of R² and R³, and R⁶ and R⁷, independently, canjoin together to form a cyclic moiety. The cyclic moiety can contain 5or 6 ring members and can be cycloalkyl, heterocycloalkyl, aryl, orheteroaryl. For example, when the cyclic moiety formed by joining R² andR³ is a benzene, it fuses with the two pyridine rings co which R² and R³are bonded, and the benzene ring and the two pyridine rings togetherform phenanthroline.

As used herein, alkyl is a straight or branched hydrocarbon chaincontaining 1 to 6 carbon atoms. Examples of alkyl include, but are notlimited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,tert-butyl, and hexyl.

By “cycloalkyl” is meant a cyclic alkyl group containing 3 to 8 carbonatoms. Some examples of cycloalkyl are cyclopropyl, cyclopentyl,cyclohexyl, cycloheptyl, adamantyl, and norbornyl. Heterocycloalkyl is acycloalkyl group containing 1-3 heteroatoms such as nitrogen, oxygen, orsulfur. Examples of heterocycloalkyl include piperidine, piperazine,tetrahydropyran, tetrahydrofuran, and morpholine.

In this disclosure, aryl is an aromatic group containing 6-12 ring atomsand can contain fused rings, which may be saturated, unsaturated, oraromatic. Examples of an aryl group include phenyl and naphthyl.Heteroaryl is aryl containing 1-3 heteroatoms such as nitrogen, oxygen,or sulfur. Examples of heteroaryl include pyridyl, furanyl, pyrrolyl,thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl, benzofuranyl, andbenzothiazolyl.

Note that an amino group can be unsubstituted, mono-substituted, ordi-substituted. It can be substituted with groups such as alkyl,cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. Halo refers tofluoro, chloro, bromo, or iodo.

A labile ligand (i.e., X¹ or X²) refers to a group which coordinateswith less affinity to the metal ion (i.e., M) of a complex of formula(I) relative to the four pyridyl nitrogen atoms. Such ligand cantherefore undergo rapid equilibrium with other labile ligands. Examplesof a labile ligand include H₂O, Cl, trifluoroacetate, or pyridine.

A metal complex of formula (I) possesses unexpectedly high specificitytoward nucleic acid containing a bulge structure. As described above, anucleic acid with such a structure is associated with various disorders.A metal complex of formula (I) can therefore be used in a diagnostic kitfor detecting nucleic acid bulge-associated disorders.

Other features or advantages of the present invention will be apparentfrom the following detailed description of several embodiments, and alsofrom the appending claims.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts the sequence of each of nucleic acid substrates A-D usedin Examples 1-3 below.

DETAILED DESCRIPTION OF THE INVENTION

The invention features metal complexes of formula (I) which specificallytarget bulge structures in a nucleic acid molecule Metal complexes offormula (I) can therefore be used in detecting nucleic acid bulges fordiagnostic purposes. A method of specifically cleaving a nucleic acidbulge using a metal complex of formula (I) is also within the scope ofthis invention.

A number of methods can be used to prepare the metal complexes offormula (I). For example, when each of L¹ and L² is —N(R^(c))— whereR^(c) is H, the hexaazacyclophane car be formed by reacting2,9-diamino-1,10-phenanthroline with 2,9-dichloro-1,10-phenanthrolinethe presence of nickel (II) ion, which can then be removed by usingtrifluoroacetic acid. See, e.g., Chang et al., J. Chin. Chem. 43, 73-75(1996).

Alternatively the method described above can be modified as shown insteps i, ii, and iii of the following scheme:

Reagents and conditions i, CH₃L K₃Fe(CN)₆/NaOH(aq); ii, PCl₅, POCl₃, 75%iii, NH_(3(g)), 200° C. 80%, iv, Co(OAc)₂ in TFA/MeOH/CH₂Cl₂, reflux,64%.

As shown in step iv above, the desired metal ion, e.g., cobalt(II), canbe coordinated to the hexaazacyclophane ligand at the same time as twoaxial ligands, e.g., trifluoroacetate, are coordinated to the metal ion.Substituents on the aromatic rings can be introduced either before orafter the preparation of the ligand by methods familiar to one ofordinary skill in the art, e.g., electrophilic aromatic substitution.

Complexes of formula (I) where each of L¹ and L² is —S— can be preparedin an analogous way by reacting starting materials such as2,9-dichloro-1,10-phenanthroline in H₂S gas instead of ammonia gas (seestep iii of the above scheme). On the other hand, a complex of formula(I) where each of L¹ and L² is —O— or —C(R^(a)) (R^(b))— can be preparedby reacting compounds such as 1,10-phenanthroline-2,9-diboronic acid and2,9-dihydroxy-1,10-phenanthroline in the presence of a palladiumcatalyst such as Pd(PPh₃)₄.

Note that the metal ion of each of the complexes of formula (I) adoptsan octahedral coordination. For example, the X-ray crystal structure ofcobalt(II)(hexaazacyclo-phane)(trifluoroacetate)₂, i.e., Co^(II)(HAPP)(TFA)₂, reveals that the complex contains two labile axial TFAligands, and two linked 1,10-phenanthroline moieties where all fourpyridyl nitrogen atoms are locating on the same coordination plane. Theaverage Co-N distance is approximately 1.86 ÅEPR spectrum of the Co^(II)complex gave a g_(av) value at 2.005-2.331 in methanol, indicating thepresence of an octahedral Co^(II) complex. When one equivalent ofpyridine was added, it rapidly displaced one of the axial TEA ligandsunder ambient conditions, as monitored by EPR spectroscopy, suggestingthat the TFA ligands are labile. The TFA ligands can also be readilysubstituted by water upon dissolution of the complex in aqueous buffer.

Due to the steric hindrance brought about by its octahedral structure, ametal complex of formula (I) does not intercalate in between bases of anucleic acid molecule. Using Co^(II)(HAPP) (TFA)₂ as an example, atopoisomerase I assay conducted in the absence of H₂O₂ and undernon-cleavage conditions (vide infra) showed no sign of DNA unwindingresulted from DNA intercalation. Further, under the same non-cleavageconditions, a native gel mobility shift assay conducted using theCo^(II) complex also showed no indication of the presence ofhigh-molecular-weight bands attributable to the presence of aDNA-Co^(II) complex adduct in polyacrylamide gel electrophoresis. Inaddition, the melting temperature of calf thymus DNA (60 μM pernucleotide) incubated with the Co^(II) complex (8 μM) only changed by0.5-1.0° C. In contrast, incubation of DNA with ethidium bromide, aknown DNA intercalator, resulted in a DNA-ethidium bromide adduct with amelting temperature differing by 12-13° C. from the control underidentical reaction conditions. Moreover, the DNA-binding constant of theCo^(II) complex, as determined by spectral titration at 399 nm with calfthymus DNA was found to be 10-fold less when compared to another knownDNA-intercalator, Cu^(II)(HAPP)⁺², which adopts a planar structure.Because of its non-intercalating nature, a metal complex of formula (I)can unambiguously detect the location of a bulge in a nucleic acid.

In the presence of H₂O₂ (0.005%-0.05%), a metal complex of formula (I)cleaves a nucleic acid molecule containing a bulge catalytically.Although the metal complex can still effect nucleic acid cleavage in theabsence of H₂O₂, a longer reaction time (about 8-10 times longer) and ahigher concentration of the metal complex (about 10-fold higher) arerequired to produce the same amount of cleavage. When H₂O₂ is replacedby magnesium monoperoxyphthaiic acid or oxone, no significant nucleicacid cleavage was observed under the same reaction conditions and time.Further, the amount of nucleic acid cleavage was reduced by half when ahydroxyl radical scavenger was added. See Example 1 below. Thisindicates that hydroxyl radicals are responsible for the nucleic acidcleavage.

Moreover, the metal complex targets nucleic acid with high specificity.Not only does the complex cleave specifically at the bulge structure,the size of such a structure also controls the specificity of thecleavage reaction. It was unexpectedly found that in treating adouble-stranded DNA substrate containing a three-base bulge and asix-base hairpin loop with Co^(II)(HAPP) (TFA)₂, cleavage occurredspecifically at the bulge, and only weakly at the loop. See Example 1below. As hydroxyl radicals are diffusible and generally lackspecificity towards a particular nucleotide or a group of nucleotides,the high specificity must have resulted from a specific recognitionbetween the metal complex and the bulge structure. Indeed, when thejust-described nucleic acid was denatured, no specific cleavage wasobserved at the sequence corresponding to the bulge. See Example 2below.

Without further elaboration, it is believed that one of ordinary skillin the art can, based on the description herein, utilized parts or thewhole procedure to its full extent. The following examples are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever. All publicationsmentioned above are incorporated by reference in their entirety.

EXAMPLE 1

λ-Phage FC-174 DNA was purchased from Life Technologies (Gibco BRL). Nofurther purification was needed prior to use. The synthetic DNAsubstrate employed herein was a 27-mer DNA,5′-GCACATCTGAGCCTGGGAGCTCTCTGC-3′ (SEQ ID No. 1) which was purchasedfrom Perkin Elmer Inc. (see nucleic acid substrate A in FIG. 1), and waspurified by gel purification in a 20% denaturing polyacrylamide gel (7 Murea). The DNA bands were visualized with an UV lamp (λ_(max)=254 nm) byplacing the gel on a TLC F254 plate (20×20 cm, Merck). After asuccessive process of excising the desired visible bands, extracting theDNA from gel, and precipitating it by EtOH, a pure DNA was obtained. TheDNA concentration was determined using the extinction coefficient(λ_(max)=260 nm) or molecular weight method (1 OD=about 33 mg and theaverage molecular weight of one nucleotide=330 daltons).

The 5′-³²P-end labeled DNA substrate was prepared by using T4polynucleotide kinase (New England Biolabs) anddeoxyadenosine-5′-[γ-³²P]-triphosphate (Amersham). The excess freeγ-³²P-ATP was removed by filtration with Centricon-10 (Amicon) usingultracentrifuge (6,000 rpm, Beckman GS-15R equipped with rotor F0850) at4° C. for 80 minutes, followed by an additional centrifuge with Milli-Qwater (1 mL) for 60 minutes. A further dilution to proper radiationintensity with deionized water was performed prior to use in assaysdescribed below.

Using the 5′-³²P-end labeled DNA substrate, a modified Maxam-Gilbert GLane was prepared by cooling a 20 μL solution containing about 10 nCi³²P-labeled substrate in deionized H₂O to 0-4° C. prior to the additionof dimethyl sulfate. The solution containing the labeled DNA was thenvortexed (<1 sec), and 2-mercaptoethanol (10 μL) was immediatelyadded tothe solution. The solution was vortexed for an additional 30 seconds.Afteradding to sonicated calf thymus DNA (5 mg) and 3.0 M sodium acetate(pH 7.0, 15 μL) to the solution, the labeled DNA was precipitated with95% EtOH and centrifuged to obtain a pellet which was then treated withpiperidine as described above prior to use as control in a DNA cleavageassay.

In the DNA cleavage assay, a 20 μL solution containing a finalconcentration of about 8 nCi of 5′-γ-³²P-labeled substrate (4-5 μM) andunlabeled DNA (4 mM) in 10 mM sodium phosphate buffer (pH 6.96) werecombined with Co^(II)(HAPP) (TFA)₂ (0.6 μM) and H₂O₂ (0.005-0.05%) at25° C. for 5 minutes. The reaction was quenched by adding sonicated calfthymus DNA (4 mg), 3 M sodium acetate (5 μL, pH 4.5), and 95% EtOH (700μL), and then stored at −78° C. for 20 minutes, centrifuged (12,000 rpm)at 4° C. for 20 minutes, and finally lyophilized to dryness to form apellet. The reaction mixture was then subjected to a piperidinetreatment by adding 0.7 M piperidine aqueous solution (60 μL) andheating at 90° C. for 30 minutes. After the reaction mixture waslyophilized, washed with deionized H₂O, and lyophilized again todryness, it was resuspended in a gel-loading buffer (5 μL) containing0.25% bromophenol blue, 0.25% xylene cyanol FF, and 7 M urea. The DNAfragment was analyzed by 20% denaturing polyacrylamide gel (7 M urea)and then visualized using Kodak BioMax MR-1 films with intensifyingscreens. The optical density of DNA fragments was quantified using imageprograms from NIH image (free shareware) and UVP Inc.(GelBase/GelBlotTMPro) equipped with an Vista S-12 scanner (UMAX).

The DNA substrate employed in this example contains a three-base bulgeand a six-base hairpin loop (see nucleic acid substrate A in FIG. 1).This DNA sequence was designed based on the RNA hairpin from thetrans-activation response element (TAR-RNA). After piperidinetreatment,the strand scission was unexpectedly found to occur specifically at thebulge (T₆, C₇, and T₈) and only very weakly at the hairpin loop(C₁₃-A₁₈). Note that both the bulge and the loop contain the same5′-CTG-3′ sequence. Minor cleavage was also found at the sites near theflanking junctions of these nucleotides. Further, no significantoxidative cleavage was observed at the 5′-GGG-3′ region in the DNAhairpin loop which have been reported to be susceptible to oxidativecleavage due to its low reduction potential. When Pt(terpy) (HET)⁺(HET=2-hydroxyethylenethiol), a known DNA intercalator which targets DNAbulges, was added to the reaction, competitive inhibition was observedand the amount of cleavage at the bulge was found to reduce remarkably.

In the absence of H₂O₂, the reaction required a higher concentration ofthe Co^(II) complex (>50 μM) as well as a longer reaction time (>40minutes) to afford the same amount of DNA cleavage at the bulge.Moreover, when magnesium monoperoxyphthalic acid (MMPP) and oxone(KHSO₅) were used instead of H₂O₂, no significant DNA cleavage wasobserved. Since the addition of superoxide dismutase and D₂O into thereaction medium did not reduce the concentration of circular DNA (FormII) formed in the DNA cleavage products mediated by this Co^(II)complex, superoxide and singlet oxygen species are not involved in thisprocess. Further, when mannitol, a hydroxyl radical scavenger, was addedinto the DNA cleavage assay medium, the amount of circular DNA (Form II)was found to be reduced by half.

The results described above showed that (1) Co^(II) (HAPP) (TFA)₂specifically cleaves DNA bulge, and (2) the cleavage reaction iseffected by hydroxyl radicals produced by the reaction of the Co^(II)complex with H₂O₂.

EXAMPLE 2

A 26-mer 5′-GCAGACTGAGCCTGGGAGCTCTCTGC-3′ (SEQ ID No.4) (D, FIG. 1) wasused as the DNA substrate. It was prepared according to the sameprocedures as described in Example 1. Note that substrate D only differsfrom substrate A in that its bulge contains one less base.

Co^(II)(HAPP) (TFA)₂ (0.6 μM) was added to substrate D under the samecleavage reaction conditions as described in Example 1 above. Enhancedand specific cleavage activity was observed at T₇ (in the bulge region).The cleavage was found to be inhibited by Pt(terpy)(HET)⁺.

EXAMPLE 3

Co^(II)(HAPP)(TFA)₂ (0.6 μM) was allowed to react under identicalconditions as described above with a single-stranded 16-mer of thesequence 5′-GCCAGATCTGAGCCTG-3′ (SEQ ID No. 2) (B, FIG. 1) in thepresence of H₂O₂. No specific cleavage was observed at the 5′-TCT-3′site, even when the concentration of the cobalt complex was increased by20-fold. The single-stranded substrate was then allowed to anneal with acomplementary DNA strand 5′-CAGGGCTCTCTGCC-3′ (SEQ ID No. 3) to form adouble-stranded DNA with a three-base bulge (C, FIG. 1). When theCo^(II) complex was added to the double-stranded substrate, enhanced DNAcleavage was observed at the 5′-TCT-3′ bulge. These results indicatethat the Co^(II) complex serves as a DNA bulge-specific cleavage reagentwithout significant specificity towards the corresponding sequence inthe single-stranded DNA.

OTHER EMBODIMENTS

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. For example, a metal complex of formula (I) can be used toeffect cleavage at a nucleic acid substrate with a hairpin loop of 1-5bases. Thus, other embodiments are also within the claims.

4 1 27 DNA Homo sapiens 1 gcagatctga gcctgggagc tctctgc 27 2 17 DNA Homosapiens 2 gccagatctg agccctg 17 3 14 DNA Homo sapiens 3 cagggctctc tgcc14 4 26 DNA Homo sapiens 4 gcagactgag cctgggagct ctctgc 26

What is claimed is:
 1. A metal complex of the following formula:

wherein each of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸, independently, ishydrogen, alkyl, alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl,amino, aminoalkyl, alkylcarbonylamino, alkylaminocarbonyl,alkylcarbonyl, alkylcarbonylalkyl, alkoxycarbonyl, alkylcarbonyloxy,cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl, orheteroaralkyl; each of R² and R³, and R⁶ and R⁷, independently,optionally joining together to form a cyclic moiety fused with the twopyridyl rings to which R² and R³, or R⁶ and R⁷ are bonded; the cyclicmoiety, if present, optionally being substituted with alkyl, alkoxy,hydroxyl, hydroxylalkyl, halo, haloalkyl, amino, aminoalkyl,alkylcarbonylamino, alkylaminocarbonyl, alkylcarbonyl,alkylcarbonylalkyl, alkoxycarbonyl, alkylcarbonyloxy, cycloalkyl,heterocycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl; each ofL¹ and L², independently, is —C(R⁸) (R^(b))—, —O—, —S—, or —N(R^(C))—;each of R^(a), R^(b), and R^(C), independently, is hydrogen, alkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, orheteroaralkyl; M is a Co, Ni, Ru, Rh, Mn, Os, Ag, Cr, Zn, Cd, Hg, Re,Ir, Pt, or Pd ion; and each of X¹ and X², independently, is a labileligand; or a salt thereof.
 2. The metal complex of claim 1, wherein eachof R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸, independently, is hydrogen,alkyl, or alkoxy.
 3. The metal complex of claim 1, wherein each of R²and R³, and R⁶ and R⁷, independently, join together to form a cyclicmoiety; the cyclic moiety being benzene.
 4. The metal complex of claim3, wherein the cyclic moiety is unsubstituted.
 5. The metal complex ofclaim 4, wherein each of R¹, R⁴, R⁵, and R⁸, independently, is hydrogen,alkyl, or alkoxy.
 6. The metal complex of claim 5, wherein each of R¹,R⁴, R⁵, and R⁸, independently, is hydrogen.
 7. The metal complex ofclaim 6, wherein each of L¹ and L², independently, is —N(R^(c))— whereR^(C) is hydrogen.
 8. The metal complex of claim 7, wherein M is Co. 9.The metal complex of claim 8, wherein X¹ and X², independently, istrifluoroacetate.
 10. The metal complex of claim 9, wherein said complexis cobalt(II) (hexaazacyclophane) (trifluoroacetate).
 11. The metalcomplex of claim 1, wherein each of L¹ and L², independently, is —S— or—N(R^(c))—.
 12. The metal complex of claim 11, wherein each of L¹ andL², independently, is —N(R^(c))— where RC is hydrogen.
 13. The metalcomplex of claim 1, wherein M is Co, Ru, or Mn.
 14. The metal complex ofclaim 13, wherein M is Co.
 15. The metal complex of claim 1, wherein X¹and X², independently, is H₂O, Cl, trifluoroacetate, or pyridine. 16.The metal complex of claim 15, wherein X¹ and X², independently, istrifluoroacetate.